1. Tomas Funda EMG postdoc seminar, Nov 6, 2013 Foto: Skogforsk

2. Tree Improvement scheme www.for.gov.bc.ca/hre/forgen

3.  Phenotypic selection www.for.gov.bc.ca/hre/forgen Tree Improvement scheme

4.  Evaluation of genetic quality www.for.gov.bc.ca/hre/forgen Tree Improvement scheme

5.  Seed orchard establishment www.for.gov.bc.ca/hre/forgen Tree Improvement scheme

6. Seed orchards Artificial populations of genetically superior trees Mass production of improved seed Assumptions Random mating No gene flow from outside

7. Genetic gain  Parental breeding values  Reproductive success  Pollen contamination  Inbreeding depression  Number of parents involved in mating (including contamination)  Reproductive success  Co-ancestry  Inbreeding Genetic diversity Foto: John Marshall Seed orchard crops’ quality

8. Seed supply from seed orchards in Sweden Overall 71% Norway spruce 62% Scots pine 83% Mature seed orchard Newly established seed orchard Site after preparation for new seed orchard

9. Foto: Kalamalka Forestry Centre, B.C. Pollen contamination Reduces genetic gain May cause maladaptation of progenies More than 50% pollen contamination detected in Swedish seed orchards www.psmicrographs.co.uk Scots pine pollen grain

10. Reducing pollen contamination Several methods tested  pollen dilution zones  buffer stands  larger orchards  supplemental pollination  phenological isolation bloom delay (cooling by water mist) different geographic region Generally low success! Foto: Kalamalka Forestry Centre, B.C. Foto: Western Forest Products Ltd. B.C.

11. Isolation tents Scots pine seed orchard “Västerhus”  ca 15 km east of Örnsköldsvik  established 1991  13.7 ha  28 replicated parents Västerhus seed orchard‘s location Foto: Skogforsk

12. Isolation tents Scots pine seed orchard “Västerhus”  ca 15 km east of Örnsköldsvik  established 1991  13.7 ha  28 replicated parents Target area for Västerhus seed © Google

13. Isolation tents – design 10 experimental units 6 tents (3 treatments × 2 replicates) tent (no further treatment) tent + fan tent + supplemental pollination 4 controls close to tents (centre) at the edges Foto: Skogforsk Tent + fan Foto: Skogforsk Tent + supplemental pollination Provided by Skogforsk

14. One ramet of 4 parents (AC3056, Y3012, Y3014 and Z2081) sampled in each experimental unit Extra pollen from 5 parents (AC1006, AC4221, Z3029, Z4003, Z4022) applied via SMP in two tents Isolation tents – design 1:1 C1:3 T+F1:4 T+P1:2 T2:3 T+F2:2 T2:1 C2:4 T+PC_26 CC_103 C 1AC305614Y301427AC305640Y410352Y301264AC301577Y301490AC3056103AC3040131Z2081 2 15Y301228Z208141AC306553AC305665AC305678 91Z2081104Z2081132Y3012 3Y301416AC305629Y301242Z208154Y410366Y301279AC305692Y3014105AC3056133Y3014 4AC107517 30AC305643Y301455Z403267Z208180 93 106Y3014134Z2081 5AC305618AC306531Z403244Y301256AC305668Y301481Z208194Y3012107Y3012135AC3056 6Y301419AC304032Y301445 57Y301269Y301282Y301495AC3040108AC3056136Y3012 7Z208120AC107533AC305646Z208158AC304070AC305683Y301296AC3015109Z2081137AC3040 8AC305621AC305634Y301247Y301459AC301571AC305684AC305697AC3056110Y4103138AC3065 9Y301222Z208135Z208148Y301260 72AC107585AC304098Y3012111AC3056139 - 10AC304023AC304036Y301449AC305661Z208173Y301486Y301299Z2081112Y3014140Y4103 11AC301524Y301237Y301250Z403262AC305674Z208187 100Y4103113Z2081141AC1075 12Y301225AC305638 51AC304063Y301475Z208188AC3015101AC1075114AC3056142Z2081 13AC305626Z403239Z2081 144AC305676Y301489Z2081102 115Y3012143Y3012

15. Questions Do the plastic tents effectively isolate trees from background pollen sources? What is the effect of each treatment on genetic diversity of resulting seed crops? Does the limited number of fathers in tents result in higher rates of self-fertilization? selfing Foto: Skogforsk pollen contamination pollen leak Foto: Skogforsk

16. Parentage determination Seed collection & extraction Germination DNA isolation Microsatellite genotyping Simple paternity exclusion Not complete match with any candidate father pollen contamination Complete match with a candidate father within-orchard mating Father the same as mother self-fertilization Father different from mother – outcrossing 1408 seeds analyzed 9 microsatellite markers combined multilocus exclusion probability 0.9994

17. Experimental unit / Treatment Contaminating seeds count Number of analyzed seeds Pollen contamination [%] 1.2 T01400.0 2.2 T01240.0 1.3 T+F01430.0 2.3 T+F01500.0 1.4 T+P01460.0 2.4 T+P01600.0 Tents08630.0 1.1 C61424.2 2.1 C61464.1 C_26 C81296.2 C_103 C81286.3 Controls285455.1 Pollen contamination in tents pollen contamination × ×

18. Experimental unit / Treatment Pollen leak count Total analyzed seeds Pollen leak [%] 1.2 T01400.0 2.2 T01240.0 1.3 T+F11430.7 2.3 T+F01500.0 1.4 T+P21461.4 2.4 T+P11600.6 Tents48630.5 Pollen leak into tents pollen leak

19. Experimental unit / Treatment Pollen leak count Total analyzed seeds Pollen leak [%] 1.2 T01400.0 2.2 T01240.0 1.3 T+F11430.7 2.3 T+F01500.0 1.4 T+P21461.4 2.4 T+P11600.6 Tents48630.5 Pollen contamination Pollen leak Experimental unit / Treatment Contamin. seeds count Total analyzed seeds Pollen contam. [%] 1.2 T01400.0 2.2 T01240.0 1.3 T+F01430.0 2.3 T+F01500.0 1.4 T+P01460.0 2.4 T+P01600.0 Tents08630.0 1.1 C61424.2 2.1 C61464.1 C_26 C81296.2 C_103 C81286.3 Controls285455.1 Control Tent Heat sum accumulation tent vs. control Tents closed Tents open

20. Experimental unit / Treatment Contaminating seeds count Total analyzed seeds Pollen contamination [%] 1.2 T01400.0 2.2 T01240.0 1.3 T+F01430.0 2.3 T+F01500.0 1.4 T+P01460.0 2.4 T+P01600.0 Tents08630.0 1.1 C61424.2 2.1 C61464.1 C_26 C81296.2 C_103 C81286.3 Controls285455.1 Pollen contamination in controls

21. Experimental unit / Treatment Contaminating seeds count Total analyzed seeds Pollen contamination [%] 1.2 T01400.0 2.2 T01240.0 1.3 T+F01430.0 2.3 T+F01500.0 1.4 T+P01460.0 2.4 T+P01600.0 Tents08630.0 1.1 C61424.2 2.1 C61464.1 C_26 C81296.2 C_103 C81286.3 Controls285455.1 Pollen contamination in controls

22. Genetic diversity of seed crops

23. TentAC1006AC4221Z3029Z4003Z4022Total SMP Total analyzed SMP success 1.4 T+P9141523218214656.2 2.4 T+P232012172910116063.1 Total323427405018330659.8 Foto: Skogforsk

24. Selfing May lead to inbreeding depression  reduction in economic value of forests (timber production, …) Fertilization of ovules by pollen from the same tree

25. Selfing Lstiburek and El-Kassaby (2010) Minimum inbreeding seed orchard design Factors affecting selfing Distance between replicates of the same parent Number of parents Fecundity Parental representation Reproductive phenology …

26. Selfing Treatment Count of selfed seeds Analyzed seeds Selfing [%] 1.2 T1514010.7 2.2 T4312434.7 1.3 T+F3114321.7 2.3 T+F5115034.0 Tents14055725.1 1.4 T+P141469.6 2.4 T+P121607.5 Tents SMP263068.5 All tents16686319.2 1.1 C21421.4 2.1 C21461.4 C_26 C121299.3 C_103 C61284.7 Controls 22545 4.0 2:3 T+F parent composition Y3012 AC3056 Y4103 Z4032 AC3056 Y3012 AC3040 AC3015 × Z2081 AC3056 Y3014 AC3056 Foto: Skogforsk

27.  Too early to make a conclusion …  The isolation tents fully eliminated pollen contamination from the studied Scots pine seed orchard  The effective number of father (N e ) was very low in the tents  Selfing was too high in the tents  Supplemental mass pollination (SMP) substantially improved seed crops in terms of both N e and selfing Summary / Conclusion Foto: Skogforsk © Google

28. Next steps  Seed crop from 2012 and possibly 2013  Fine scale assessment of mating dynamics in the tents  Cost-benefit evaluation of the tent isolation method Foto: Anders Fries

29. Xiao-Ru Wang Curt Almqvist Ulfstand Wennström Bengt Andersson Jin Pan Baosheng Wang Takeshi Torimaru Financial support Föreningen Skogsträdsförädling

30.  Too early to make a conclusion …  The isolation tents fully eliminated pollen contamination from the studied Scots pine seed orchard  The effective number of father (N e ) was very low in the tents  Selfing was too high in the tents  Supplemental mass pollination (SMP) substantially improved tent seed crops in terms of both N e and selfing Summary / Conclusion Foto: Skogforsk © Google